Toxoplasmosis is a widespread infectious disease in man and animals, with variable prevalence in different countries, depending on socio-economic habits and climate. The seroprevalence of Toxoplasma gondii (T. gondii) infection is up to 95% in some populations especially in areas with hot, humid, climates and lower altitudes; and lower in colder areas. This is because moist, warm soil is favorable for T. gondii oocysts which are able to survive for about 1 year (Remington et al., Toxoplasmosis. In: Infectious diseases of the fetus and newborn infant. 7th edition. Edited by Remington J S, Klein J O, Wilson C B, Nizet V, Maldonado Y A. Pennsylvania: Elsevier Saunders; 2011: 915-1041). It has been reported that T. gondii seroprevalence in women at child-bearing age (1990-2000) was 51-72% in several Latin-American countries, 58% in Central European countries, and 54-77% in West African countries. Low seroprevalences of 4 to 39% were reported in southwest Asia, Korea and China as well as in cold and arid climate areas such as Scandinavian countries (11-28%). Meanwhile in the USA, a seroprevalence of 9.0% was recorded for 12-49 year old people during 1999-2004 (Nutter et al., JAMA 2004; 229: 1394-1398). In Malaysia seroprevalence among 200 pregnant women at University Malaya Medical Centre in 2003 was reported to be ˜49%, and the highest prevalence was in Malays, followed by Indians (Nissapatorn & Abdullah, Southeast Asian J Trop Med Public Health. 2004 March; 35(1): 24-30).
Toxoplasma can be transmitted to humans by three main routes i.e. (i) ingestion of oocysts released by infected cats in their feces. Humans are exposed to oocysts from cat litter or from soil as a result of activities like gardening or eating unwashed fruits or vegetables; (ii) ingestion of raw or inadequately cooked infected meat; and (iii) a newly infected pregnant woman passing the infection to her unborn fetus, causing congenital toxoplasmosis. In addition, immunocompromised patients such as those with AIDS and those undergoing organ transplantation are at risk of reactivated or primary infection.
Congenital toxoplasmosis is one of the most important manifestations of the infection. Acute infections in seronegative pregnant women can be transmitted to the fetus and cause severe illnesses e.g. abortion, intracranial lesions, mental retardation, retinochoroiditis, blindness, and epilepsy. An estimated 400-4,000 cases of congenital toxoplasmosis occur each year in the United States (Lopez et al., MMWR Recomm Rep. 2000 Mar. 31; 49(RR-2): 59-68); and 9% of those children have significant visual impairment (Tan et al., Am J Ophthalmol 2007; 144: 48-653). The global annual incidence of congenital toxoplasmosis is estimated to be 190,100 cases, and it is equivalent to 1.20 million DALYs, with high burdens reported in South America, some Middle Eastern countries and low-income countries (Torgerson & Mastroiacova, Bull World Health Organ. 2013 Jul. 1; 91(7): 501-8). It was estimated that the expense of a patient with severe congenital toxoplasmosis was about USD 1 million (Remington et al., Toxoplasmosis. In: Infectious diseases of the fetus and newborn infant. 7th edition. Edited by Remington J S, Klein J O, Wilson C B, Nizet V, Maldonado Y A. Pennsylvania: Elsevier Saunders; 2011: 915-1041).
A panel of tests is usually performed on serum samples of persons with suspicion of infection. This includes testing for IgM and IgG and performing IgG avidity assays. Usually, specific IgM appears 1 week after infection and IgG appears 1 to 3 weeks later (Jenum et al., J Clin Microbiol 1998; October; 36(10): 2907-13). Thus, anti-T. gondii IgM is usually the first assay performed; or both IgM and IgG assays are performed at the same time. If both are positive, then IgG avidity test is often performed; low IgG avidity indicates that the infection may be acute whereas high IgG avidity confirms it is a chronic infection.
The availability of a highly sensitive IgM test is important. Currently, there is no reference Toxoplasma IgM test. One of the more sensitive tests for IgM detection is ISAGA (immunosorbent agglutination assay), a micro agglutination plate assay produced in France (BioMereieux); however it is not frequently used in developing countries due to its high cost. The most common serological test format in routine diagnosis of toxoplasmosis is ELISA. Major diagnostic laboratories use ELISA kits adapted for expensive fully automated systems such as VIDAS® (BioMerieux, France), ARCHITECT™ (Abbot, USA) and LIAISON® (DiaSorin, USA). The result using these advanced systems is usually quite reliable. Nevertheless, even the detection of IgM in newborns using these advanced systems is still a challenge as evidenced in a recent report that showed only 3 of 10 cases of newborns with confirmed congenital toxoplasmosis were detected using LIAISON® and VIDAS® (Murat et al., Expert Rev Anti Infect Ther 2013 September; 11(9): 943-56). Thus there is still a need for development of a highly sensitive and highly specific test for detection of specific IgM to indicate T. gondii infection using well-defined antigens.
The host-pathogen interaction during infectious disease is complex, multifaceted and dynamic. All regulated virulence factors of a pathogen cannot be determined only by using in vitro methods, since it is impossible to reproduce all the various environmental stimuli that occur at the site of the actual infection. Genes induced only or at greater level during in vivo growth (as compared to in-vitro growth) may be virulence determinants and are thus important in the natural pathogenesis process (Valdivia and Falkow, Science, 1997; 277: 2007-2011 Angelichio and Camilli, Infection and Immunity 2002; 70: 6518-6523). These in vivo-induced proteins have good potential as diagnostic markers, vaccine candidates, therapeutic targets and in further understanding of the pathogenesis of the pathogen (Handfield et al., Trends Microbiol 2000; 8: 336-39; John et al., Infection and Immunity 2005; 73: 2665-79; Hongwei et al., 2009; Lowry et al., Plos One, 2011; 6 (3): e17425).
To identify the in vivo induced proteins, several techniques have been used such as signature-tagged mutagenesis (STM) (Hensel et al., Science, 1995; 269: 400-403), in vivo expression technology (IVET) (Angelichio and Camilli, Infection and Immunity 2002; 70: 6518-6523), differential fluorescence induction (DFI) (Valdivia and Falkow, Science, 1997; 277: 2007-2011) and transcriptional and proteomic profiling (Ramachandran et al., Science 2004; 305: 86-90). Although these techniques have contributed significantly to expand the understanding of pathogen pathogenesis, they have some important limitations. Most notably they require an animal model to monitor the pathogen mechanism which may not adequately mimic events in an actual site of infection within the natural human host.
In vivo-Induced Antigen Technology (IVIAT), a modification of previously described immunologic screening techniques of protein expression libraries, has been introduced to identify antigens expressed specifically in vivo during natural human infection (Handfield et al., Trends Microbiol 2000; 8: 336-39). IVIAT has been used with the same goals as STM, IVET and DR yet does not need the use of animal models. IVIAT is preferred to those other techniques since it directly examines human immune responses by utilizing sera from patients infected with a pathogen of interest, thus reducing false-positive results caused by the differences between proteins expressed during actual human infection and in-vitro conditions (Handfield et al, Trends Microbiol 2000; 8: 336-39). So far, IVIAT has successfully been applied to the study of over 30 different prokaryotic and eukaryotic organisms. We have previously used IVIAT to identify a number of in vivo-induced antigens in chronic and acute human toxoplasmosis (Amerizadeh et al., Microb Pathog. 2013a; 54: 60-66; Amerizadeh et al., BMC Infectious Disease 2013b; 13: 287-299). Although three antigens were induced 54-330 fold in vivo over in vitro levels, the sensitivity and specificity for detecting T. gondii-IgM was only 90% and 40-70%, respectively.
There is a need to identify T. gondii antigens that are highly induced in vivo compared to in vitro and which have high sensitivity and specificity for detecting T. gondii-IgM, as they would not only be useful for diagnosing toxoplasmosis but would also be potential vaccine candidates.
There is currently no vaccine for human Toxoplasma infection. Vaccines for animal infection are available but can be improved. There is also a need to improve treatment for toxoplasmosis, especially for congenital infection.